CN109705203B - Protein related to plant type and coding gene and application thereof - Google Patents

Abstract

The invention discloses a protein related to plant types and a coding gene and application thereof. The protein related to the plant type disclosed by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is the protein of sequence 1; A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function; A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). Experiments prove that the protein related to the plant type and the gene thereof can regulate the plant type, particularly the plant height of the plant, the expression of the gene can be inhibited to obviously reduce the plant height of the plant, the protein related to the plant type and the gene thereof can be used for cultivating the plant with reduced plant height, and have important application value for effectively regulating and controlling the plant type of the plant through genetic breeding and genetic engineering methods.

Description

Protein related to plant type and coding gene and application thereof

Technical Field

The invention relates to a protein related to plant types, a coding gene and application thereof in the field of biotechnology.

Background

Rice, as an important food crop, lives over one third of the world's population. With the growing population, the reduction of the cultivated land area and the deterioration of the environment, rice production is under greater and greater pressure in terms of ensuring yield. The plant type of rice is an important agronomic trait related to yield, and mainly depends on several aspects of plant height, leaf type, tillering number, tillering angle, spike morphology and the like. Therefore, the discovery and the utilization of the related gene for controlling the rice plant type have important significance for rice breeding and production.

Cell division and expansion are the most fundamental physiological processes in plant growth and development, and plant cells complete cell proliferation by continuously forming new cell plates and cell walls, and are accompanied by a plurality of complex physiological processes in the process of division and expansion, wherein vesicle transport of an intracellular endomembrane system is responsible for the transport of a plurality of synthetic raw materials.

Endocytosis of plant cells affects not only the most basic cell differentiation function, but also plant growth and development, transmission of hormonal signals, and communication with the external environment, such as nutrient transport, tolerance to toxic substances, resistance to pathogenic bacteria, and the like. The clathrin-mediated endocytosis is the most main route of plant cell endocytosis, and the clathrin initially forms vesicles on cell membranes, recognizes and wraps specific endocytic goods on the cell membranes, and then transports substances into cells. Compared with extensive and intensive research on animal cells, the research on the endocytosis mechanism of plant cells at present is mainly based on the research model of animal cells and the hypothesis of related theories, and the plant cells still have great research space due to the existence of the division mechanism which is different from that of the animal cells.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the plant type of a plant, particularly the plant height.

In order to solve the technical problems, the invention firstly provides any one of the following applications of the plant type related protein derived from rice or a substance for regulating the activity or the content of the plant type related protein;

D1) regulating and controlling plant types of plants;

D2) preparing a plant type regulating product;

D3) cultivating short-stalk plants;

D4) preparing and cultivating short-stalk plant products;

the plant type related protein (the name of which is DGSP1) is A1), A2) or A3 as follows:

A1) the amino acid sequence is the protein of sequence 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing may be labeled as shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The DGSP1 protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 1 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The DGSP1 protein in A2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding DGSP1 protein in A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID NO.2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end a coding sequence of the tag shown in the above table. Wherein, the DNA molecule shown in the sequence 2 encodes DGSP1 protein shown in the sequence 1.

The invention also provides any one of the following uses of the biological material related to DGSP 1;

D1) regulating and controlling plant types of plants;

D2) preparing a plant type regulating product;

D3) cultivating short-stalk plants;

D4) preparing and cultivating short-stalk plant products;

the biomaterial is any one of the following B1) to B40):

B1) a nucleic acid molecule encoding DGSP 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell containing the nucleic acid molecule of B1);

B10) a transgenic plant cell containing the expression cassette of B2);

B11) a transgenic plant cell containing the recombinant vector of B3);

B12) a transgenic plant cell containing the recombinant vector of B4);

B13) transgenic plant tissue comprising the nucleic acid molecule of B1);

B14) transgenic plant tissue comprising the expression cassette of B2);

B15) transgenic plant tissue containing the recombinant vector of B3);

B16) transgenic plant tissue containing the recombinant vector of B4);

B17) a transgenic plant organ containing the nucleic acid molecule of B1);

B18) a transgenic plant organ containing the expression cassette of B2);

B19) a transgenic plant organ containing the recombinant vector of B3);

B20) a transgenic plant organ containing the recombinant vector of B4);

B21) a nucleic acid molecule that reduces the expression level of DGSP 1;

B22) an expression cassette comprising the nucleic acid molecule of B21);

B23) a recombinant vector comprising the nucleic acid molecule of B21);

B24) a recombinant vector comprising the expression cassette of B22);

B25) a recombinant microorganism comprising the nucleic acid molecule of B21);

B26) a recombinant microorganism comprising the expression cassette of B22);

B27) a recombinant microorganism containing the recombinant vector of B23);

B28) a recombinant microorganism containing the recombinant vector of B24);

B29) a transgenic plant cell containing the nucleic acid molecule of B21);

B30) a transgenic plant cell containing the expression cassette of B22);

B31) a transgenic plant cell containing the recombinant vector of B23);

B32) a transgenic plant cell containing the recombinant vector of B24);

B33) transgenic plant tissue comprising the nucleic acid molecule of B21);

B34) transgenic plant tissue comprising the expression cassette of B22);

B35) transgenic plant tissue containing the recombinant vector of B23);

B36) transgenic plant tissue containing the recombinant vector of B24);

B37) a transgenic plant organ containing the nucleic acid molecule of B21);

B38) a transgenic plant organ containing the expression cassette of B22);

B39) a transgenic plant organ containing the recombinant vector of B23);

B40) a transgenic plant organ containing the recombinant vector of B24).

In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) or B15):

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b12) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b13) a DNA molecule shown in a sequence 3 in a sequence table;

b14) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) or b13) and encoding DGSP 1;

b15) a cDNA or DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) or b14) and encodes DGSP 1;

B21) the nucleic acid molecule may be a DNA fragment represented by formula I below:

SEQ forward-X-SEQ reverse (I);

the SEQ forward direction is a partial fragment of sequence 2 or the full length thereof;

the sequence of the SEQ reverse direction is complementary to the sequence of the SEQ forward direction in a reverse direction;

and the X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to the SEQ forward direction and the SEQ reverse direction in sequence.

In the above application, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The SEQ forward direction may specifically be the nucleotide sequence at position 400-799 of SEQ ID NO 2.

The nucleotide sequence of the DNA fragment shown in the formula I is a sequence 4 in a sequence table, the nucleotide sequence from the 1 st to the 400 th site in the sequence 4 is a nucleotide sequence from the 400 th-through 799 th site in the sequence 2, the nucleotide sequence from the 429 th-through 1616 th site is a nucleotide sequence of Arabidopsis FAD2 intron, and the nucleotide sequence from the 1623 th-through 2045 th site is a nucleotide sequence which is reversely complementary to the nucleotide sequence from the 400 th-through 799 th site in the sequence 2.

The nucleotide sequence encoding DGSP1 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of DGSP1 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode DGSP1 protein and have the function of DGSP1 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown as coding sequence 1 shown as sequence 2 or 3 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding DGSP1 protein (DGSP1 gene expression cassette) described in B2) refers to a DNA capable of expressing DGSP1 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of DGSP1 gene, but also a terminator that terminates transcription of DGSP1 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter T7lac, a constitutive promoter of cauliflower mosaic virus CaMV35S, a tomato ribulose-1, 5-bisphosphate carboxylase minor (rbcs) gene promoter; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: t7 terminator, Agrobacterium tumefaciens nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (1985), Nature, 313: 810; Rosenberg et al (1987), Gene,56: 125; Guerineau et al (1991), mol.Gen.Genet,262: 141; Propudfoot (1991), Cell,64: 671; Sanfacon et al, GenesDev., 5: 141; Mogen et al (1990), plantatcell, 2: 1261; Munroe et al (1990), Gene, 91: 151; Ballad et al (1989), nucleic acids, Res.17: 7891; Joshi et al (1987), Resclei, 9615: 9627).

The recombinant vector containing the DGSP1 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pET-28a, pCAMBIA2301, pSP72, pROKII, pBin438, pCAMBIA1302, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co., Ltd.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

The existing RNA interference vector can be used for constructing a recombinant vector containing a DNA molecule for reducing the expression level of DGSP1, such as pLHRNAi.

B23) The recombinant vector can be pLHRNAi-DGSP 1. The pLHRNAi-DGSP1 is a recombinant vector obtained by replacing a DNA sequence between SacI and SnaBI recognition sites (recognition sequences) of pLHRNAi with a DNA fragment shown in a sequence 4.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. The bacteria can be derived from Escherichia (Escherichia), Erwinia (Erwinia), Agrobacterium (Agrobacterium) such as Agrobacterium tumefaciens EHA105, Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

In the above application, the plant type may be plant height.

In the above application, the plant may be M1) or M2) or M3) or M4):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) plants of the genus oryza;

m4) rice.

The invention also provides any one of the following methods:

x1) to obtain a target plant with reduced plant height compared with the acceptor plant, wherein the method comprises the steps of reducing the content of DGSP1 in the acceptor plant, or reducing the activity of DGSP1 in the acceptor plant, or inhibiting the expression of a gene coding for DGSP1 in the acceptor plant;

x2), which comprises reducing the content of DGSP1 in a receptor plant, or reducing the activity of DGSP1 in the receptor plant, or inhibiting the expression of a coding gene of DGSP1 in the receptor plant, so as to obtain a target plant with reduced plant height compared with the receptor plant, and realize the reduction of the plant height.

The recipient plant contains the gene encoding DGSP 1.

In the above method, inhibiting the expression of a gene encoding DGSP1 in a recipient plant can be achieved by introducing B21) said nucleic acid molecule into said recipient plant.

B21) Said nucleic acid molecule can be obtained by introducing a recombinant vector containing the nucleic acid molecule according to B21) into said recipient plant.

B21) The nucleic acid molecule can be realized by introducing the recombinant vector of B23) or B24) into the recipient plant.

In the method, the encoding gene of DGSP1 can be modified as follows and then introduced into a receptor plant to achieve better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the amino acid sequence of the gene encoding DGSP1 of the present invention may be changed to conform to plant preferences while maintaining the amino acid sequence, depending on the preferred codons of the recipient plant; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding DGSP1 can be introduced into a recipient plant by using a recombinant expression vector containing the gene encoding DGSP 1.

Both the recombinant vector and the recombinant vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition)).

The plant of interest is understood to comprise not only the first generation plants in which the DGSP1 protein or the gene encoding it has been altered, but also the progeny thereof. For the plant of interest, the gene may be propagated in the species, or transferred into other varieties of the same species, including commercial varieties in particular, using conventional breeding techniques. The plant of interest includes seeds, callus, whole plants and cells.

In the above method, the recipient plant may be M1) or M2) or M3) or M4):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) plants of the genus oryza;

m4) rice.

The invention also provides the following products of Y1) or Y2):

y1) B21) the nucleic acid molecule;

y2) a product for regulating plant type, said product comprising DGSP1 or said biological material.

The product for regulating plant type of the plant can take DGSP1 or the biological material as an active ingredient, and can also combine DGSP1 or the biological material with substances with the same functions as the active ingredient.

In the product, the plant type can be the plant height.

In the above product, the plant may be M1) or M2) or M3) or M4):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) plants of the genus oryza;

m4) rice.

Experiments prove that the plant type related protein and the gene thereof can regulate and control the plant type, particularly the plant height of the plant. Inhibiting the expression of the plant type related protein gene can obviously reduce the plant height of the plant, and proves that the plant type related protein and the gene thereof play an important role in regulating and controlling the plant height of the plant. The invention not only provides a basis for further clarifying the molecular mechanism of plant types of plants, but also provides new gene resources and breeding resources for plant breeding. The plant type related protein, the gene thereof and the nucleic acid molecule for inhibiting the gene expression can be used for cultivating plants with reduced plant height; the transgenic plant with the reduced DGSP1 gene expression, which is obtained by the invention, can be used as a new plant seed material for researching the molecular mechanism of plant dwarfing and finding more genes for regulating and controlling the plant height development of plants. The invention has important application value for effectively regulating and controlling the plant type of the plant by utilizing the gene resource through genetic breeding and genetic engineering methods.

Drawings

FIG. 1 shows the transcript level detection of DGSP1 gene in wild type rice and transgenic interfering families.

FIG. 2 is a phenotypic observation of RNA interference transgenic rice with reduced expression level of DGSP1 gene.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

The rice Kitaake (also called wild-type rice, abbreviated WT) in the following examples is described in GaoH, ZhengXM, WanJM., et al, Ehd4encodes a Novel and Oryza-gene-specific regulator of photo-technological flowability in rice PLOS GENET.2013,9(2): e1003281) which is publicly available from the institute of crop science of the Chinese academy of agricultural sciences, and is used only for repeating the experiments related to the present invention and is not applicable for other uses.

The expression vector pLHRNAi used in the following examples is pLHRNAi in Chinese patent 201110055864.X, and the public can obtain the biological material from the research institute of crop science of Chinese academy of agricultural sciences, and the biological material is only used for repeating the related experiments of the invention and cannot be used for other purposes.

The Agrobacterium used in the examples described below was Agrobacterium tumefaciens EHA105(Agrobacterium tumefaciens EHA105) (New Agrobacterium hel plasmids for gene transfer to plants. hood, ElizabethE; Gelvin, Stanton B; Melchers, LeoS; Hoekema, Andre. transfer research,2(4): p.208-218(1993)), which was publicly available from the research of crop science of the Chinese academy of agricultural sciences, and which was used only for the repetition of the experiments related to the present invention and was not used for other purposes.

Example 1 plant type-related protein DGSP1 can regulate and control the plant height of rice

The embodiment provides a plant type related protein derived from rice Kitaake (Oryza sativa var. Kitaake), which is named as DGSP1, wherein the amino acid sequence of DGSP1 in the rice Kitaake is sequence 1 in a sequence table, the coding sequence (namely CDS sequence) of DGSP1 gene is sequence 2 in the sequence table, and the genome sequence of DGSP1 gene is sequence 3 in the sequence table.

Construction of RNA interference vector for reducing DGSP1 gene expression level

1. Obtaining of DGSP1 gene expression interference fragment

(1) Total RNA of 14-day seedlings of rice Kitaake (Oryzasativa) was extracted using an RNAprep plant total RNA extraction kit (Tiangen Biochemical technology, Beijing) Co., Ltd.) and reverse-transcribed to obtain cDNA.

(2) And (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and a primer pair consisting of DGSP1-sense-F and DGSP1-sense-R to obtain a fragment 1(SEQ forward direction).

DGSP1-sense-F：5'-CTAGGTACCAGGCCTGAGCTCGCGTTAGGTAGGAACATTG-3'；

DGSP1-sense-R：5'-GACGTAGGGGCGATAGAGCTCGAGCTTCTAATGATACCCC-3'。

(3) And (2) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and a primer consisting of DGSP1-antisense-F and DGSP1-antisense-R to obtain a fragment 2(SEQ reverse direction).

DGSP1-antisense-F:5'-TCTTAGAATTCCCGGGGATCCGCGTTAGGTAGGAACATTG-3'；

DGSP1-antisense-R:5'-CGTTACGTAGTCGACGGATCCGAGCTTCTAATGATACCCC-3'。

2. Construction of DGSP1 Gene RNA interference vector (recombinant expression vector pLHRNAi-DGSP1)

(1) The expression vector pLHRNAi is digested by restriction endonuclease SacI to obtain a linear expression vector pLHRNAi, and the linear fragment is recovered. Integrating the fragment 1 obtained in the step 1 (2) to a linear expression vector pLHRNAi (the concrete method refers to a clone infusion kit (please ensure the white) instruction) by adopting a homologous recombination directional cloning method to obtain a homologous recombination product 1, then transferring the homologous recombination product 1 to an escherichia coli DH5 alpha competent cell, culturing overnight at 37 ℃, and marking the obtained recombination vector with a correct sequence as pLHRNAi-sense-DGSP 1.

(2) The recombinant vector pLHRNAi-sense-DGSP1 is cut by restriction endonuclease SnaBI enzyme to obtain a linear vector pLHRNAi-sense-DGSP1, and the linear vector is recovered. Integrating the fragment 2 obtained in the step 1 (3) onto a linear vector pLHRNAi-sense-DGSP1 by adopting a homologous recombination directional cloning method (the concrete method refers to the clone infusion kit instruction), obtaining a homologous recombination product 2, transferring the homologous recombination product 2 into an escherichia coli DH5 alpha competent cell, culturing overnight at 37 ℃, and marking the obtained recombinant vector with a correct sequence as pLHRNAi-DGSP 1.

(3) Sequencing is carried out on the recombinant vector pLHRNAi-DGSP1, and the result shows that the recombinant vector pLHRNAi-DGSP1 is formed by inserting a double-stranded DNA fragment shown by a nucleotide sequence from 400 th to 799 th bit from 5 'end of SEQ ID No.2 into the SacI enzyme cutting site of the expression vector pLHRNAi in the forward direction, inserting a double-stranded DNA fragment which is reversely complementary to the double-stranded DNA fragment shown by the nucleotide sequence from 400 th to 799 th bit from 5' end of SEQ ID No.2 into the SnaBI enzyme cutting site, thus successfully replacing the DNA sequence between the SacI and the SnaBI recognition sites (recognition sequences) of the pLHRNAi with a DNA fragment shown by a sequence 4 and a reverse complementary DNA fragment thereof, and connecting the two DNA fragments by partial DNA fragments in the vector.

Constructing RNAi interference transgenic plant with reduced DGSP1 gene expression level and identifying transgenic plant

One) construction of transgenic plants

The rice Kitaake is mediated and transformed by a recombinant vector pLHRNAi-DGSP1 through Agrobacterium tumefaciens EHA105, and an empty vector control plant is constructed by utilizing the expression vector pLHRNAi according to the same method, and the specific method is as follows:

1. and (3) introducing the recombinant vector pLHRNAi-DGSP1 obtained in the step one into the Agrobacterium tumefaciens EHA105 by a heat shock method to obtain the recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector pLHRNAi-DGSP 1. The recombinant Agrobacterium tumefaciens EHA105 was cultured at 28 ℃ for 16 hours, and the cells were collected. The bacterial cells were diluted with N6 liquid medium (Sigma, catalog No. C1416) containing 100. mu.M acetosyringone to obtain diluted bacterial solution, OD600 of which was about 0.5.

2. Mixing mature embryogenic callus of rice Kitaake cultured for one month with the diluted bacterial solution obtained in step 1, infecting for 30min, drying the surface bacterial solution of callus by filter paper, transferring into N6 solid co-culture medium (N6 mixed medium formula is potassium nitrate (2800mg/L), ammonium sulfate (463mg/L), potassium dihydrogen phosphate (400mg/L), magnesium sulfate (MgSO 2)₄·7H₂O) (185mg/L), calcium chloride (CaCl)₂·2H₂O) (165mg/L), disodium ethylene diamine tetraacetate (37.3mg/L), ferrous sulfate (FeSO)₄·7H₂O) (27.8mg/L), manganese sulfate (MnSO)₄·H₂O) (4.4mg/L), zinc sulfate (ZnSO)₄·7H₂O) (1.5mg/L), boric acid (1.6mg/L), potassium iodide (0.8mg/L), vitamin B1 (thiamine hydrochloride) (1.0mg/L), vitamin B6 (pyridoxine hydrochloride) (0.5mg/L), nicotinic acid (0.5mg/L), glycine (2.0mg/L), sucrose (20000 mg/L). Weighing 24.1g of N6 mixed culture medium, heating and stirring to dissolve in 1000ml of distilled water, adjusting pH to 5.8 with sodium hydroxide, and autoclaving at 115 ℃ for 20 minutes to obtain N6 solidAnd (5) culturing the culture medium. ) Co-culturing at 24 deg.C for 3d to obtain co-cultured callus.

3. The callus after the co-culture treatment in step 2 was inoculated on N6 solid selection medium (a medium obtained by adding hygromycin to N6 solid selection medium, the mass concentration of hygromycin in the N6 solid selection medium being 150mg/L) containing hygromycin at a mass concentration of 150mg/L for the first selection.

4. And (3) picking the healthy callus on the 16 th day from the first screening, transferring the healthy callus to an N6 solid screening culture medium (a culture medium obtained by adding hygromycin to an N6 solid culture medium, wherein the mass concentration of the hygromycin in the N6 solid screening culture medium is 200mg/L) containing the hygromycin, culturing, and carrying out secondary screening, wherein the secondary screening is carried out once every 15 days and is carried out for 1 time in total, and the obtained healthy callus is the resistant callus.

5. And (3) selecting the resistant callus obtained in the step (4), transferring the resistant callus to a differentiation medium (the differentiation medium: 6-BA 2mg, NAA 0.2mg and N6 mixed medium is 4g, hydrolyzed casein is 1g, inositol is 0.1g, cane sugar is 25g, sorbitol is 2.4g, agar powder is 7g, deionized water is supplemented to 1L) containing hygromycin with the mass concentration of 150mg/L, carrying out differentiation culture, culturing for 45d at 24 ℃ (the height of the overground part of the plant is about 15cm), opening a bottle mouth, hardening seedlings for 3 days, and then transplanting to a greenhouse for culture, namely a transferred pLHRNAi-DGSP1 plant (T0 generation).

II), RNAi interference transgenic plant PCR identification with reduced DGSP1 gene expression level

Extracting genome DNA of T0 generation seedlings of the transferred pLHRNAi-DGSP1 plant and seedlings of the rice Kitaake plant (abbreviated as WT) obtained in the step one), and performing PCR molecular detection by using primers 1390-F (5'-TGCCTTCATACGCTATTTATTTGC-3') and FAD2-R (5'-GAAGCGACGGACCTGGAGAT-3') to identify positive seedlings, wherein the obtained plants of 562bp PCR products are positive transgenic plants, and the rice Kitaake plant (wild type) cannot obtain 562bp PCR products. Two positive transgenic plants are taken and named as a pLHRNAi-DGSP1 transferred plant RNAi-1 (RNAi-1 plant for short) and a pLHRNAi-DGSP1 transferred plant RNAi-2 (RNAi-2 plant for short) respectively.

Third, RNAi interference transgenic plant with reduced DGSP1 gene expression level identification of DGSP1 gene expression level

Respectively extracting RNA of the RNAi-1 plant (interference family 1), the RNAi-2 plant (interference family 2) and the rice Kitaake plant (wild type) leaf obtained in the second step, setting internal reference as ubiptin, and carrying out fluorescence quantitative PCR by using internal reference primers UBI-F and UBI-R and DGSP1 gene specific quantitative primers DGSP1-qRT-F and DGSP1-qRT-R to detect the change of the expression level of the DGSP1 gene of each plant. The results (fig. 1) show that the expression levels of the DGSP1 gene in RNAi-1 and RNAi-2 plants were significantly reduced compared to the expression level of the DGSP1 gene in rice Kitaake (wild type), and that there was no significant difference in the expression levels of the DGSP1 gene in the empty vector control plant and rice Kitaake. The primers are as follows:

UBI-F:5’-GCTCCGTGGCGGTATCAT-3’；

UBI-R:5’-CGGCAGTTGACAGCCCTAG-3’；

DGSP1-qRT-F:5’-GATTGGTCGCAATGCAGTAGCC-3’；

DGSP1-qRT-R:5’-GATTGGTCGCAATGCAGTAGCC-3’。

RNAi interference transgenic plant phenotype identification with reduced DGSP1 gene expression level

And (3) respectively planting the RNAi-1 plant (interference family 1), the RNAi-2 plant (interference family 2), the empty carrier control plant and the rice Kitaake plant (wild type) obtained in the step two in a sequential experimental base of the institute of crop science of the Chinese academy of agricultural sciences, and observing the phenotypic difference of each plant in the whole growth period. The observation results are shown in fig. 2, compared with the rice Kitaake (wild type) plant, the RNAi-1 (interfering family 1) plant and the RNAi-2 (interfering family 2) plant both have the phenotype of plant height dwarfing, the average plant heights of the rice Kitaake (wild type), the RNAi-1 (interfering family 1) plant and the RNAi-2 (interfering family 2) plant are 69.61 +/-3.53 cm, 43.33 +/-2.16 cm and 38.42 +/-1.73 cm respectively in the rice mature period, and the heights of the empty vector control plant and the rice Kitaake plant are not obviously different. Thereby proving that the DGSP1 gene participates in controlling the growth and development of rice plant types.

<110> institute of crop science of Chinese academy of agricultural sciences

<120> plant type-related protein, and coding gene and application thereof

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 570

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Asp Arg Leu Arg Ala Gly Ser Pro Val Tyr Gly Arg Gln Arg Ser

1 5 10 15

Gly Ser Ser Thr Gly Ser Ser Ser Pro Gly Gly Val Ser Pro Ser His

20 25 30

His Arg Ser Ser Ser Thr Ser Ser Ala Ala Ser Ala Ala Ala Gly Leu

35 40 45

Gly Gly Gly Val Ser Asn Val Arg Arg Thr Gln Asn Val Ala Ala Arg

50 55 60

Ala Ala Ala Ala Arg Leu Ala Gln Val Met Ala Ser Gln Ser Ala Ala

65 70 75 80

Ala Ala Ala Gly Arg Asp Asp Asp Asp Asp Asp Asp Asp Tyr Ala Asn

85 90 95

Asp His Pro Pro Ala Pro Pro Pro Ala Arg Phe Gly Ser Ala Arg Pro

100 105 110

Ala Ala Ala His Gly Ser Asn Gly Val Ser Leu Leu Gly Arg Thr Ala

115 120 125

Arg Ser Pro Ser Pro Ala Leu Gly Arg Asn Ile Val Glu Pro Pro Pro

130 135 140

Thr Val Arg Ser Thr Ser Ala Gly Arg Pro Ala Val Ala Ser Arg Pro

145 150 155 160

Thr Thr Thr Val Val Pro Pro Ile Lys Thr Ser Thr Thr Leu Arg Thr

165 170 175

Pro Ser Pro Ile Pro Pro Val Ala Val Glu Pro Pro Val Asp Arg Ser

180 185 190

Arg Gln Lys Arg Phe Asp Thr Gly His Leu Asn Ser Arg Glu Ser Thr

195 200 205

Pro Lys Arg Glu Ala Ser Ala Leu Gln Asp Glu Leu Asp Ile Leu Gln

210 215 220

Glu Glu Asn Glu Ser Val Leu Glu Lys Leu Arg Leu Ala Glu Glu Arg

225 230 235 240

Cys Glu Glu Ala Glu Ala Arg Ala Lys Glu Leu Glu Lys Gln Val Ala

245 250 255

Ala Leu Gly Glu Gly Val Ser Leu Glu Ala Arg Leu Leu Ser Arg Lys

260 265 270

Glu Ala Ala Leu Lys Gln Arg Glu Ala Ala Leu Lys Ala Ala Arg Glu

275 280 285

Ser Lys Asp Gly Lys Asp Gly Glu Val Thr Thr Leu Lys His Glu Leu

290 295 300

Asp Cys Ala Lys Glu Glu Val Val Thr Ala Met Glu Gln Leu Lys Glu

305 310 315 320

Ala Glu Thr Glu Thr Lys Ala Leu Arg Ser Met Thr Gln Arg Met Ile

325 330 335

Leu Thr Gln Glu Glu Met Glu Glu Val Val Leu Lys Arg Cys Trp Leu

340 345 350

Ser Arg Tyr Trp Gly Leu Ala Val Gln Tyr Gly Val Tyr Pro Glu Ile

355 360 365

Ala Val Ser Lys His Glu His Trp Ser Ser Leu Ala Pro Leu Pro Leu

370 375 380

Glu Val Val Leu Ser Ala Gly Gln Lys Ala Lys Glu Glu Pro Leu Lys

385 390 395 400

Gln Gly Glu Asp Asp Ala Gln Arg Arg Asn Lys Leu Val Arg Asp Met

405 410 415

Ser Asp Val Met Gly Glu Gly Asn Ile Glu Ser Met Leu Ser Val Glu

420 425 430

Met Gly Leu Arg Glu Leu Ser Ser Leu Lys Val Glu Asp Ala Val Val

435 440 445

Val Ala Leu Gly Gln His Arg Arg Pro Ser Ile Val Arg Gln Phe Thr

450 455 460

Ser Asp Phe Lys Ser Pro Gly Glu Pro Lys Phe Leu Glu Ala Phe Asp

465 470 475 480

Leu Ser His Glu Glu Ala Glu Asp Val Ser Phe Lys Gln Ala Trp Leu

485 490 495

Ile Tyr Phe Trp Arg Arg Ala Lys Thr His Gly Ile Glu Glu Asp Ile

500 505 510

Ala Glu Glu Arg Leu Gln Phe Trp Ile Gly Arg Asn Ala Val Ala Pro

515 520 525

Thr Ser His Asp Ala Ile Asp Val Glu Arg Gly Leu Thr Glu Leu Arg

530 535 540

Lys Leu Gly Ile Glu Gln Gln Leu Trp Glu Gly Ser Arg Ala Asp Ile

545 550 555 560

Asp Glu Asp Ser Ser Ala Ile Glu Asn His

565 570

<210> 2

<211> 1713

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

atggaccgcc tccgcgcggg gagccccgtc tacgggcggc agcggagcgg cagcagcacg 60

ggctcctcct ccccgggcgg cgtctccccg tcccaccacc gctcctcctc cacctcctcc 120

gccgcctccg ccgccgcggg gctgggcggc ggcgtctcca acgtgcgccg cacgcagaac 180

gtcgcggcgc gggcggccgc cgcgaggctg gcccaggtca tggcgtcaca gagcgccgcg 240

gccgccgcgg gccgcgacga cgacgacgac gacgacgact acgccaatga ccacccgccc 300

gcccctcccc ccgcgaggtt cggctccgcg cgccccgccg cggcgcacgg cagcaacggc 360

gtctcgttgc tcggccgcac cgcgagatct ccctcccctg cgttaggtag gaacattgta 420

gagccacctc ctactgtccg ctcaacatca gctgggaggc ctgccgttgc ttcacggccg 480

accaccacgg tggttccacc gatcaaaacc agcacgacat tgcggacacc atcgcctatt 540

ccacctgtgg ctgtggagcc tccagtggac cgcagtcggc agaaaaggtt tgatacaggg 600

catctcaact ccagagaatc aacaccaaaa agggaggcgt ctgcacttca agatgagctt 660

gatatactac aagaggagaa tgagagtgtt ctagaaaagc tacgacttgc tgaagaaaga 720

tgtgaagaag cagaagctag agccaaggag cttgagaaac aggtagctgc ccttggggaa 780

ggggtatcat tagaagctcg cctcttgagc aggaaagaag ctgcccttaa acagagggag 840

gctgcactga aggctgcaag ggaatcaaaa gatggtaaag atggggaagt gacaacacta 900

aagcatgaac tggattgtgc caaagaagag gttgtaacag ctatggagca gctaaaggaa 960

gcagagactg aaacaaaggc cctccggtcc atgactcaga gaatgatctt aacccaagag 1020

gaaatggaag aggtggtcct taaaaggtgc tggctttcac gttattgggg cttagcggtt 1080

caatacggag tttatcccga gattgcggta tcaaagcatg aacattggtc atcattagct 1140

cctcttcccc ttgaggttgt tctctctgct ggtcaaaagg ctaaggagga acctctcaaa 1200

caaggtgagg atgacgccca aaggagaaat aaacttgtgc gagatatgag tgatgtaatg 1260

ggagaaggca atatagagag catgctctca gttgaaatgg ggcttagaga gctttcatca 1320

ttgaaggtgg aagatgctgt tgtggttgca cttggtcaac accggagacc tagtatagtt 1380

cggcaattca catcagattt taaatcacct ggtgaaccta aattcttgga ggcgtttgat 1440

cttagccatg aagaggcaga agatgttagc tttaagcagg catggcttat atacttctgg 1500

agaagagcta aaactcatgg cattgaggaa gacattgctg aagaacggct tcagttctgg 1560

attggtcgca atgcagtagc cccaacttct catgatgcta tagatgtgga gcgaggtcta 1620

acagagctca ggaaactggg catagagcag caactgtggg aaggatcacg tgcagatatc 1680

gatgaagatt catcggcaat tgagaatcac tag 1713

<210> 3

<211> 7123

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

acctccttcc cacttctcaa cctcgcctcg ccatctccgc gccgcggccc agatccctcc 60

ggcgagcgcc gccgccgcca ccgcctccgc catggaccgc ctccgcgcgg ggagccccgt 120

ctacgggcgg cagcggagcg gcagcagcac gggctcctcc tccccgggcg gcgtctcccc 180

gtcccaccac cgctcctcct ccacctcctc cgccgcctcc gccgccgcgg ggctgggcgg 240

cggcgtctcc aacgtgcgcc gcacgcagaa cgtcgcggcg cgggcggccg ccgcgaggct 300

ggcccaggtc atggcgtcac agagcgccgc ggccgccgcg ggccgcgacg acgacgacga 360

cgacgacgac tacgccaatg accacccgcc cgcccctccc cccgcgaggt tcggctccgc 420

gcgccccgcc gcggcgcacg gcagcaacgg cgtctcgttg ctcggccgca ccgcgagatc 480

tccctcccct gcggtaaatt ctgcttcccc cggatctctc gctgatctcg ctacccgctg 540

cgtctcaact taggatttcg atggtttgaa ctgtcgcggt gcacgaatga gtcatagttg 600

gtcaatcgga agtcaaattg gttctgaagc atgcttgagc agatgaattt gctagtgaag 660

ctagtttaca tctcgccaat gtcagtccaa ttctatagcg atgccttctg aattgtaaaa 720

attgggagaa gtgatgaatg cattgggact tctggctgct tcgtgatttg atcaccgctt 780

atcctgtgcc cttctgctaa tttgatctcc tcatgttttc attagttagg taggaacatt 840

gtagagccac ctcctactgt ccgctcaaca tcagctggga ggcctgccgt tgcttcacgg 900

ccgaccacca cggtggttcc accgatcaaa accagcacga cattgcggac accatcgcct 960

attccacctg tggctgtgga gcctccagtg gaccgcagtc ggcagaaaag gtacgtcaat 1020

gacattcccc agatctgttg catggtctgc agcattctct gtgttttcta tacttaaaat 1080

agcattcctt aagttctact tctcttagga tatattaaaa tgaatgcatg gttaattgta 1140

ctagcaaccc caccaaatcg agcagaaatg agttattttt actgcttgcg ctgtttttca 1200

gaagaagtga tgttggtagt tcacaacatt aactaaactg ctcttgataa agggatgtat 1260

ttggttggtg gcgttgttag taaaaaaaga tgcattgata tcataaggtt gaaccaacag 1320

ccctatgcca aactatgtgt tcagttttgg tcggtatagt ggagagccct cttatcttgg 1380

gcgtaatgga ttgaaaagtt ttgagttcaa taatacatgc ttattaagga tgggtagtaa 1440

attattatgc gtccgaaatc ttaatcgctt acctaaattg tgaaatctga tatttgattg 1500

aatcaattgg tcagcatctt ggggttgtgg gtgtgcctct tggccagttt cctattaccg 1560

cgctttgctg aactgcgccg cagaatctat caaattatac ataattttta cctattctag 1620

caaatgattt cttttattgt gctgaaaagg gcattgctga ctagttatta ataataacaa 1680

aaaattcggc cccaagggga aagatctccc ctgaggtatt tcaattaaga agaagcacct 1740

gaaaccgagg ccccagaaag gccacaaaag ctggtcctcc tacatgggga gccgccccct 1800

atggatcggg ccttaatcca tgctttgagg ctcttgcccc ctacacaagg acgatagatc 1860

aattcctaac cttgttttgg tagaaccagc gagtgacctt ttttggtgca agaccaagat 1920

ttgaaccctg gtcgttagct tcccactgga aggtatttac catcatgcta caagcacgtt 1980

tacttgctga ctagttaata tatctaggat tagtaaaata aagtgccctt tagtcgtcca 2040

ccatgctaaa cgcacccttt tctcttttaa gtatgttact aggggcacca cattgcttgt 2100

tgcattaaag ttttggaata atcacattat gaccatctgg ccacttctta tagcttgaat 2160

ttctttttcc cattttttca aatgttccta aaaataggca tcaggtaagt tatttacttt 2220

ttttttttgc ctcaggtttg atacagggca tctcaactcc agagaatcaa caccaaaaag 2280

ggaggcgtct gcacttcaag atgaggtttt ccttattctc tataaactag ttatacgtat 2340

aatatatttt agctcatggt tctgtcacga actttgacct agaacttttt gaggagtgtt 2400

tatcagatgt ttttaagact aatgcaatgc tgtcctacag cttgatatac tacaagagga 2460

gaatgagagt gttctagaaa aggtgaaata gcaaccatag agctgtttta taggcaatct 2520

agtacttgat tttatactga tctttaacaa attattttct gaatcttgat tgacagctac 2580

gacttgctga agaaagatgt gaagaagcag aagctagagc caaggagctt gagaaacagg 2640

tgagataatc ttatattaaa cattgtttta tctttttttt tgtattatga taaattggac 2700

atgtgcacta gacattgagt ctatctctag ttctcactac agtttttctt ttggcgggaa 2760

tttttcacta caatttaaac ttgaatttga attattggaa cgaatattct cctttacttg 2820

aggaggtgtt cattacactg agcattttgt atctttggtt agctaaggtt atatttgttg 2880

atatgcaggt agctgccctt ggggaagggg tatcattaga agctcgcctc ttgagcaggt 2940

ttgcatcatg tttggatatg catgtttcac aaattcagaa ctgatcatag actcataacc 3000

taacattgtc atgattattg gcaggaaaga agctgccctt aaacagaggg aggttagttt 3060

ttcttcttgt gttttaccta tggatagctg gcatgccaat cttgtagtac tgtttttttt 3120

atcttagaat ttatatgtaa aatttggaag tgttagaatt ttagtgtttt tcttgttgtg 3180

aaaacaaaag gatgaaacat tttggaattt catttgatct tttttctgct ttatttgtct 3240

tgaaatgcac atgcggtact atagtcatgc taggttctta ttgtaaaaaa aaaatctgaa 3300

cctgaaggat atgctggagt acataccttc caattttacc agaaaaataa agaggatatt 3360

tcttacccat caacgaacac tttatataat gctttttgtc tacaggctgc actgaaggct 3420

gcaagggaat caaaagatgg taaagatggg gaagtgacaa cactaaagca tgaactggat 3480

gtaaatatac ttcctaccaa tcaaaatatg gaagtagtgt tggtttgtat atggtgtttt 3540

atttgaccat gttttcttct catgctttga atagtgtgcc aaagaagagg ttgtaacagc 3600

tatggagcag ctaaaggaag cagagactga aacaaaggcc ctccggtcca tgactcagag 3660

aatgatctta acccaagagg aaatggttag tctgatttca gtactcccat tttttaattg 3720

cttgatatag attttgatct atttgcaacc ttatgctagg aagaggtggt ccttaaaagg 3780

tgctggcttt cacgttattg gggcttagcg gttcaatacg gtaagttgct aagagtattt 3840

ctgtaagaaa tgcttttagt gttatcttgt ataagctgtt ctgtaatcat gctctctaga 3900

caaaataaaa tcaaagctta tcatgtcaaa aggatgttag ttatcatggt gaaacaaaag 3960

aaataaatta tgttgcacat ttgttatttg ttgttctctg ttgaaaggat gttagttatc 4020

atgtgattac tttgtttggt taaatatcaa tagtgtgagc tagttgctgg aaattggaaa 4080

ggcaaacatt ttttttgtga cttatatgca atttggttta ttgcgtttac agaaatggtt 4140

ttcaacatag tttttgtttc gcagtttatc tttagatgct gataattcac gttcacacta 4200

gacctgagaa tttggtctgg tctgaatgca agtttaaggc tggtttggtc tcagaaaatg 4260

aggccaatgg ccaaacagtc catactccat cctgactatg tatatggcta ggtgtcactt 4320

agattagggc ctagggggcc tgaaagtaac tagttgtgac cctggtctgg tctcgaaaat 4380

gcttaattct agtttgcaca tgatgttgaa atgtactgtt aactttttcc ctgtccgtga 4440

tttatctgat ggcccattaa taacttgaag gagtttatcc cgagattgcg gtatcaaagc 4500

atgaacattg gtcatcatta gctcctcttc cccttgaggt tgttctctct gctggtcaaa 4560

aggctaagga ggaacctctc aaacaaggca tgtctgcatc tttataatat acaagttgta 4620

gatcgtagtt tgttccatgt actccagtta attattcttg gatcttttca agcaggtgag 4680

gatgacgccc aaaggagaaa taaacttgtg cgagatatga gtgatgtaat gggagaaggc 4740

aatatagaga gcatgctctc agttgaaatg gggcttagag agctttcatc attgaaggta 4800

ttccactgcc cttttattgg cagtggttta ttaactttct gttctataga ttctcagaga 4860

tgaaattcca gacacagtac ttgtactcat tctaatgcag agacgaacat ttacagcaaa 4920

aatggcacaa cagtaattct tctaatatcc tctagagttt ttttttatgg agtgttctct 4980

gaaatacgga agtgtgaaca gtatttgcca ggctttacct tacttccact gtaatagagc 5040

actaagtaat cattcttttt gtactgagta tatttgcatg tttggacaag tgacaaacca 5100

tatgtgcttt ataaatacag gttttttttt ctaggcttcc ttatcacttc tctcctcact 5160

agttattgtt ctgtaggtgg aagatgctgt tgtggttgca cttggtcaac accggagacc 5220

tagtatagtt cggcaattca catcaggttt gcacttcttt tatcatttca tatcattctt 5280

gtttaactta catgcgtaat atgatttctt cattggtgct gccttcactc ataaaaaaaa 5340

actaagaaag gtataaattc ttctcttatg cagattttaa atcacctggt gaacctaaat 5400

tcttggaggc gtttggtaat ggttcttccc tttgtctaat agatttatgc aaaacttcca 5460

ctaacagctt gagcttgtgt tgtatgtaaa catttcatcc tgtttctatg agtttaaatt 5520

aaatttgaac tttttgacct attgcagatc ttagccatga agaggcagaa gatgttagct 5580

ttaagcaggt aaacctttca gcgatgtgaa ctatcaaatt catccattta ttacgtgtga 5640

tttatggaat atatattgaa tgcaggcatg gcttatatac ttctggagaa gagctaaaac 5700

tcatggcatt gaggaagaca ttgctgaaga acggcttcag ttctggattg gtcgcaatgc 5760

agtagcccca acttctcatg atgctataga tggtaataag caactttcct tgttgaaaaa 5820

ataacctagc attgggagtt atccacctgt tagttaatga tttatcgtac tatttaacca 5880

tccaacgtcc ctgggatgtc ttttatttgt gcagctcttt tttgacagca tgccccaaca 5940

gttgatcatg tgatgctttt gtagggatca aaccaacttt ctatttcctc cttttattat 6000

ttcttcactg ttgtatttta tctctttgcc ctttactgtc ctctttccca tctgtaatcc 6060

aaagtacggt atagtggtac gaacattatg gtcccgttcg tttctccaac aagagttgga 6120

tgaagattta gattttcgtg gcacactttt gaaaccgcta aacggtgcgt ttcgtgcgaa 6180

aactttctat atggaagttg ttttaaaata tcagattaat ccatttttca agtttgtaat 6240

aattaaaact catttaatca cacgttatta ccacatcgtt ttgcgtgaaa cacttaatct 6300

ttatcttcat cttcatcttc aggagattca aacaccacct atatgtgaga tacttacatg 6360

aaccactatt aatgtgattg atgtgttttt ttttgtctaa acagttatgg gatattgggg 6420

gtatacagaa ggcattttga gtatgccctc atatatatta catgttgcgg tgtgcctata 6480

atctgttttg tttttccaat tgtttttacc tagatttaca cacctcatcc attttttctc 6540

ctatccaaga atgccatcat tttcagtttt ttgctttact gagcactgtt agttttgtta 6600

tctgcagtgg agcgaggtct aacagagctc aggaaactgg gcatagagca gcaactgtgg 6660

gaaggatcac gtgcagatat cgatgaagat tcatcggcaa ttgagaatca ctaggcacat 6720

ctcaaggttg tttatggtac atgattattc caattttgga gaatggaaat gctttttcgc 6780

ggctggtctt gggccattgc catcattgga tccctgaatg gtgctgcaat cttcgggtgt 6840

tgagcaatgg gcagtcttcg ggtgttgagc gatattatgg gcttgtccag agatattgtg 6900

tagtgcatgt gcgagccatt tatcttttgt cgaaatattt ttgtgaaatg tgtaattgtt 6960

ttgtcctggc aatatgatgg tgatctcact tcatccaatt tgtgaatttg tgattacacc 7020

acatgtgtac tgcctgttat tcagtaaaat atacagagag gaagctatgc ttcctgcgac 7080

aaatgagcaa atcataagtg atgattcttt ctggtagtct gcc 7123

<210> 4

<211> 400

<212> DNA

<213> Rice (Oryza sativa)

<400> 4

gcgttaggta ggaacattgt agagccacct cctactgtcc gctcaacatc agctgggagg 60

cctgccgttg cttcacggcc gaccaccacg gtggttccac cgatcaaaac cagcacgaca 120

ttgcggacac catcgcctat tccacctgtg gctgtggagc ctccagtgga ccgcagtcgg 180

cagaaaaggt ttgatacagg gcatctcaac tccagagaat caacaccaaa aagggaggcg 240

tctgcacttc aagatgagct tgatatacta caagaggaga atgagagtgt tctagaaaag 300

ctacgacttg ctgaagaaag atgtgaagaa gcagaagcta gagccaagga gcttgagaaa 360

caggtagctg cccttgggga aggggtatca ttagaagctc 400

Claims (6)

1. Any one of the following applications of the substance for reducing the expression level of the plant type related protein;

D1) cultivating short-stalk rice;

D2) preparing and cultivating short-stalk rice products;

the plant type related protein is A1) or A2) as follows:

A1) the amino acid sequence is the protein of sequence 1;

A2) a1) at the N-terminus or/and the C-terminus.

2. Use of any one of the following biomaterials associated with a plant type-related protein according to claim 1;

D1) cultivating short-stalk rice;

D2) preparing and cultivating short-stalk rice products;

the biomaterial is any one of the following B1) to B20):

B1) a nucleic acid molecule for reducing the expression level of a plant type-related protein according to claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell containing the nucleic acid molecule of B1);

B10) a transgenic plant cell containing the expression cassette of B2);

B11) a transgenic plant cell containing the recombinant vector of B3);

B12) a transgenic plant cell containing the recombinant vector of B4);

B13) transgenic plant tissue comprising the nucleic acid molecule of B1);

B14) transgenic plant tissue comprising the expression cassette of B2);

B15) transgenic plant tissue containing the recombinant vector of B3);

B16) transgenic plant tissue containing the recombinant vector of B4);

B17) a transgenic plant organ containing the nucleic acid molecule of B1);

B18) a transgenic plant organ containing the expression cassette of B2);

B19) a transgenic plant organ containing the recombinant vector of B3);

B20) a transgenic plant organ containing the recombinant vector of B4).

3. Use according to claim 2, characterized in that:

B1) the nucleic acid molecule is a DNA fragment shown as the following formula I:

SEQ forward-X-SEQ reverse (I);

the SEQ forward direction is a partial fragment of sequence 2 or the full length thereof;

the sequence of the SEQ reverse direction is complementary to the sequence of the SEQ forward direction in a reverse direction;

and the X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to the SEQ forward direction and the SEQ reverse direction in sequence.

4. Use according to claim 3, characterized in that: the SEQ forward direction is the nucleotide sequence at position 400-799 of the sequence 2.

5. Any one of the following methods:

x1), reducing the expression level of the plant type-related protein in the receptor rice, or inhibiting the expression of the plant type-related protein coding gene in the receptor rice, so as to obtain the target rice with reduced plant height compared with the receptor rice;

x2), comprising the steps of reducing the expression level of the plant type related protein in the receptor rice as claimed in claim 1, or inhibiting the expression of the coding gene of the plant type related protein in the receptor rice as claimed in claim 1, so as to obtain the target rice with reduced plant height compared with the receptor rice, and realize the reduction of the plant height of the rice.

6. The method of claim 5, wherein: inhibiting the expression of a gene encoding the rice plant type-associated protein according to claim 1 in recipient rice by introducing the nucleic acid molecule according to B1) of claim 2 or 3 into the recipient rice.

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